2019.01.11: Coating and seeding
- Prepare 5 plates of 60 mm of diameter with 26 coverslips (9 mm)
- Treatment with laminin 1h (2’4 microg/ml diluted in Borate Buffer)
- Wash with PBS 1x (3 times)
- Seed aprox 20000 cells/cm2 –> every plate has 21 cm2 –> 500000 cells/plate 60 mm (in proliferation medium)
- Incubate at 37ºC
2019.01.14: proliferation medium exchange
Change the medium of the cells –> new proliferation medium
2019.01.15: treatment and medium exchange (differentiation)
Change the medium of the cells –> Add retinoic acid to the differentiation medium (1 microl RA in 1 ml differentiation medium)
*Change the half of the differentiation medium (+ retinoic acid) every 2 or 3 days –> 18.01.19 / 21.01.19
2019.01.22: ICC
Samples:
- 1-49: Small fiber PNP
- 50: negative control 1
- 51: negative control 2
- 52: JGV (positive control)
- 53: 15-198 (CNTN1+)
ICC Protocol:
- Fix with PFA 4% . Incubation at RT during 15 min.
- Wash with PBS 1x
- Block with Goat Serum 5 % (incubation at RT during 1 h)
- Patient’s sera diluted 1/100 with Goat Serum 5 %
- Wash with PBS 1x
- Secondary antibody: GAH 488 IgG diluted 1/1000 in Goat Serum 5 %
- Wash with PBS 1x
- Vectashield mounting medium
Results: las neuronas están muy mal (pocos axones y muchas células muertas.
Problema con la diferenciación –> hay que hacerlo a 10.000 cels/cm2, y añadir 1 microl de RA por cada ml de medio que hay en la placa (es decir, si cambio la mitad del medio, añadir el doble de RA).
